Epithelial cell growth promoter

ABSTRACT

It is intended to provide a novel oligopeptide which can be relatively easily produced, has not only a hair growth-stimulating effect but also an effect of promoting the growth of epithelial cells (for example, skin regeneration) and can easily pass through the horny layer to thereby reach the desired target cells in which its effects are to be exerted. Namely, water-soluble oligopeptides containing a proryl isoleucyl glycyl unit or an isoleucyl glycyl serine unit and having from 3 to 7 amino acids and water-soluble salts thereof.

This application is a 371 of PCT/JP05/05677, filed Mar. 28, 2005, whichclaims foreign priority to JP 2004-108449, filed Mar.31, 2004 and JP2004-200862, filed Jul. 7, 2004.

TECHNICAL FIELD

The present invention relates to a novel water-soluble oligopeptidehaving epithelial cell growth-promoting effect such as hairgrowth-promoting effect, skin regeneration-promoting effect, therapeuticeffect on skin ulcer, and therapeutic effect on mucosal injury. Moreparticularly, the present invention relates to a water-solubleoligopeptide and a water-soluble salt thereof comprising 3 to 7 aminoacid units containing a particular amino acid sequence and to anepithelial cell growth promoter comprising them as an active ingredient.

BACKGROUND TECHNOLOGY

Recently, the control mechanisms of hair growth and hair loss have beenelucidated, while a variety of new hair growth agents such as novelcompounds having the hair growth-promoting effect, ingredients based ongenetic research, combinations of the Chinese traditional medicinesystem and others have been proposed along with increased public concernfor hair growth agents.

FIG. 1 is an illustrative diagram showing a hair cycle that repeats hairgrowth and hair loss. A normal hair has a hair papilla 3 in a hair root2 of a hair body 1 thereof and has a trichogen cell 4 above the hairpapilla 3. The hair body 1 grows up, enters a regression phase (catagen)and stops growing in approximately 2 to 3 weeks. Then, it enters aresting phase (telogen) for 2 to 3 months. In the meantime, the hairroot 2 continues its activities and generates a new hair body 1′. Thisnew hair body 1′ proceeds to enter a growth phase (anagen) and loses theold hair body 1. It further keeps growing and regenerates an originalhair form in approximately 5 to 6 years.

Hair growth agents have the effect of promoting trichogen cell growth ineach phase of such a hair cycle, inducing the anagen phase in thetelogen phase, prolonging the anagen phase, or delaying shift to thecatagen phase and perform the promotion of hair growth or the inhibitionof hair loss.

For example, a hair growth agent comprising6-(1-piperidinyl)-2,4-pyrimidinediamine-3-oxide (minoxidil) as an activeingredient (see U.S. Pat. No. 4,139,619), a hair growth compositioncomprising 1 to 6% by mass of minoxidil, polyhydric alcohol, ethanol,pyridoxin hydrochloride, and water (see Japanese Patent Laid-Open No.2002-326913), a hair growth agent comprising a fibroblast growthfactor-10 (FGF-10) as an active ingredient (see Japanese PatentLaid-Open No. 10-279501), a pilatory comprising particular fatty acidester, ether, monoglyceride sulfate, or monoalkyl glyceryl ether sulfateas an active ingredient (see Japanese Patent Laid-Open No. 11-246359), ahair growth agent comprising a CRF1 receptor antagonist as an activeingredient (see the pamphlet of International Publication No. WO02/019975), an aerosol preparation for hair growth containing: a gelledcomposition for aerosol comprising a crude drug extract with bloodcirculation-promoting effect and vitamin or a derivative thereof asactive ingredients and having a water-soluble polymer mixed therewith;and a propellant (see Japanese Patent Laid-Open No. 7-101834) andelsewhere have previously been proposed as such hair growth agents.

Alternatively, an oral hair growth agent comprising, as an activeingredient, a peptide represented by the formula, R¹-Met-Ile-XR² (in theformula, X represents Trp, Phe, Trp-Leu, Phe-Leu, Tyr-Leu, Ile-Leu, orLeu-Leu; R¹ represents a hydrogen atom or an amino-protecting group; andR² represents a hydroxyl- or carboxyl-protecting group) or apharmacologically acceptable salt is known as a special hair growthagent (see the pamphlet of International Publication No. WO 00/29425).

In addition, information from very recent newspaper has reported therelease of a hair growth agent having 6-benzyl aminopurine (cytopurine)in combination with pentadecane (see the article issued on Mar. 2, 2004,Nikkei Business Daily).

These hair growth agents have their own advantages, and some of them areapproved to exhibit significantly effects. However, they cannotcompletely respond to all symptoms and certain raw materials thereof aredifficult to obtain so that they are not necessarily adequate forpractical applications. Therefore, the appearance of a novel hair growthagent exhibiting excellent effects has been demanded in this field.

On the other hand, a skin lotion for anti-aging consisting of apolymerization product with an average molecular weight of 280 to 20000of a tripeptide composed of one glycine residue and two other amino acidresidues in a degradation product of collagen or gelatin obtained bycollagenase (see Japanese Patent Laid-Open No. 2000-309521), acollagen-producing promoter comprising a mixture of tripeptidesconsisting of (Gly-Ala-Arg), (Gly-Ala-Hyp), (Gly-Ala-Lys),(Gly-Pro-Ala), (Gly-Pro-Arg), (Gly-Pro-Hyp), and (Gly-Pro-Ser) as anactive ingredient (see Japanese Patent Laid-Open No. 2003-137807) andelsewhere are known as physiologically active substances comprising anoligopeptide or a polymerization product thereof as an activeingredient. However, no epithelial cell growth promoter comprising atripeptide as an active ingredient has been proposed so far.

DISCLOSURE OF THE INVENTION

An object of the present invention is to provide a novel water-solubleoligopeptide that can be produced in a relatively simple way, has notonly the hair growth-promoting effect but also the epithelial cellgrowth-promoting effect such as skin regeneration, and can easily passthrough the stratum corneum to reach and act on a desired target cell.

The inventors have conducted intensive studies to develop chemicalsuseful as a hair growth agent and, as a result, have arrived at adiscovery that an active substance exhibiting excellent hair growtheffect is present in an extract from the culture supernatant of bacteriaof the genus Bacillus, that the hair growth effect results from aparticular oligopeptide structure in this active substance, and that anyparticular polypeptide having this oligopeptide structure not onlyexhibits desired hair growth effect but also exhibits the effect ofpromoting cell regeneration in skin transplantation or in therestoration of skin ulcer or aging skin. The inventors have furtherarrived at a discovery that a water-soluble oligopeptide havingparticular amino acid units exhibits excellent epithelial cellgrowth-promoting effect leading to completion of the present inventionon the basis of these discoveries.

Namely, the present invention provides a water-soluble oligopeptide anda water-soluble salt thereof having 3-7 amino acid units containing aprolyl-isoleucyl-glycyl unit or an isoleucyl-glycyl-serine unit, and anepithelial cell growth promoter comprising at least one selected fromthe water-soluble oligopeptide and the water-soluble salt thereof as anactive ingredient.

The effect of the water-soluble oligopeptide in the epithelial cellgrowth promoter of the present invention is exerted not only in theoligopeptide per se but also in a polypeptide having these oligopeptideunits as its molecular structure unit. However, those having a molecularweight of 500 or more are rendered slightly soluble in water so thatthey are not preferable as a hair growth agent.

Among such water-soluble oligopeptides, a tripeptide includesisoleucyl-glycyl-serine and prolyl-isoleucyl-glycine. And, atetrapeptide is exemplified, for example, by this tripeptide with anamino acid residue such as a glycyl, alanyl, arginyl, asparagyl, lysyl,seryl, valyl, or glutamyl group bonded to the front or rear, of whichglycyl-prolyl-isoleucyl-glycine (SEQ ID NO: 1) andprolyl-isoleucyl-glycyl-serine (SEQ ID NO: 2) are preferred.

A pentapeptide is exemplified, for example, by theglycyl-prolyl-isoleucyl-glycyl (SEQ ID NO: 1) group with an amino acidresidue such as a seryl group or a threonyl group bonded to the front orrear, of which glycyl-prolyl-isoleucyl-glycyl-serine (SEQ ID NO: 3) andglycyl-prolyl-isoleucyl-glycyl-threonine (SEQ ID NO: 4) are preferred.

Further, preferable hexapeptide and heptapeptide are any of those havingthe above-mentioned pentapeptide unit at the carboxyl terminus, such as,for example, alanyl-glycyl-prolyl-isoleucyl-glycyl-serine (SEQ ID NO:5), seryl-glycyl-prolyl-isoleucyl-glycyl-serine (SEQ ID NO: 6),glycyl-seryl-glycyl-prolyl-isoleucyl-glycyl-serine (SEQ ID NO: 7) andthe like.

The water-soluble oligopeptide of the present invention may be in a freeform or a water-soluble salt form. This water-soluble salt includes, forexample, sodium salt, potassium salt, lithium salt, ammonium salt andthe like.

The oligopeptide of the present invention can be produced by reacting araw material amino acid having the α-amino group protected and an aminoacid having the carboxyl group protected by any conventional method forforming a peptide bond in synthesis of a polypeptide such as, forexample, a condensation method, active ester method, azide method, mixedacid anhydride method and others, so as to form a peptide, which is inturn repeatedly subjected to the step of eliminating the protectinggroups.

This condensation method is the most general method for forming apeptide bond. In this method, for example, dicyclohexylcarbodiimide(DCC), diisopropylcarbodiimide (DIPC),N-ethyl-N′-3-dimethylaminopropylcarbodiimide (WSCl) and hydrochloridesthereof (WSCl.HCl), benzotriazole-1-yl-tris(dimethylamino)phosphoniumhexafluorophosphide (BOP), diphenylphosphoryl diazide (DPPA) and thelike are used alone as a condensing agent or in combination withN-hydroxysuccinimide (HONSu), 1-hydroxybenzotriazole (HOBt), or3-hydroxy-4-oxo-3,4-dihydro-1,2,3-benzotriazine (HOObt).

In the active ester method, for example, p-nitrophenyl ester (ONp),N-hydroxysuccinimide ester (ONSu), pentafluorophenyl ester (OPfp) andthe like can be used as an active ester.

The azide method is a method in which an amino acid or a peptide isreacted with anhydrous hydrazine to form a corresponding hydrazide, andis known as a segment condensation method with low racemization.

Furthermore, the mixed acid anhydride method is a method in whichisobutyloxycarbonyl chloride, diethylacetyl chloride, trimethylacetylchloride and the like can be used to form a mixed anhydride of thecarboxyl group of an amino acid, and is advantageous since it canstrongly activate the carboxyl group at low temperatures.

On the other hand, those easily eliminated by acid treatment,hydrolysis, or catalytic reduction are used as protecting groups for anamino acid. Among such protecting groups, a protecting group for anα-amino group includes benzyloxycarbonyl group, tert-butoxycarbonylgroup, 9-fluorenylmethoxycarbonyl group, 3-nitro-2-pyridinesulfenylgroup, methoxybenzyloxycarbonyl group and the like. The protection of acarboxyl group is performed with methyl or ethyl ester, benzyl ester,tert-butyl ester, phenacyl ester, or the like.

In the case of an α-amino acid having a hydroxyl group at the sidechain, this hydroxyl group has to be protected. The protecting group issuitably a benzyl group that is easily eliminated by catalytic reductionwith a platinum black catalyst or by strong acid treatment and, atert-butyl group that is easily eliminated by weak acid treatment.

Such an α-amino acid ester or a raw material amino acid with the aminogroup or hydroxyl group protected can be easily obtained as a commercialproduct.

The production of the oligopeptide of the present invention can beperformed by both of a liquid phase method wherein a raw material aminoacid or a derivative thereof is evenly dissolved in a solvent to effectthe reaction, and a solid phase method wherein a peptide chain iselongated on an insoluble resin, and is advantageously performed with anautomatic solid-phase synthesizer. According to this method, a desiredoligopeptide can be obtained in a short time and in a high purity.

The novel oligopeptide or water-soluble salt thereof of the presentinvention is obtained as a racemic body and can also be obtained,according to desire, as a substance having optical activity bysubjecting the racemic body to optical resolution by any conventionalmethod. This optical resolution can be performed by a method in which adiastereomer formed between the racemic amino acid and an appropriateoptically active substance is subjected to fractional crystallization, amethod using an enzyme, or a method performed by high-performance liquidchromatography (HPLC) using a chiral carrier.

The oligopeptide of the present invention is soluble in water or alcoholcompounds. This can be identified by mass spectrometry, an infraredabsorption spectrum, or high-performance liquid chromatography.

The oligopeptide of the present invention has the effect of directlypromoting hair bulb keratinocytes (HBK) growth or, particularly,trichogen cell growth, that is, hair growth-promoting effect. Inaddition, it has the effect of directly promoting epidermal cell growthand is thus useful in the graft of cultured skin or in the treatment ofskin ulcer and skin defective injury. Therefore, the oligopeptide of thepresent invention can be used as an epithelial cell growth promoter.

A preparation of the epithelial cell growth promoter of the presentinvention is obtained by dissolving the above-mentioned oligopeptideserving as an active ingredient in a concentration of 0.0001 to 5% bymass in an aqueous medium. A mixture solvent of water and awater-soluble organic solvent are preferable as the aqueous medium usedin this procedure.

For example, alcohol compounds such as ethyl alcohol, polyhydric alcoholcompounds such as ethylene glycol, diethylene glycol, dipropyleneglycol, glycerin, and 1,3-butylene glycol, polar organic solvents suchas dimethylformamide and dimethylsulfoxide are used as the water-solubleorganic solvent. These can be used alone or can be used as a combinationof two kinds or more. A preferable aqueous solvent is a mixture solventof water, propylene glycol, and ethyl alcohol.

It is optional according to desire that this epithelial cell growthpromoter of the present invention contains other compounds having thehair growth-promoting effect such as, for example, minoxidil, carproniumchloride, glyceryl pentadecanoate, tocopherol acetate, piroctoneolamine, glycyrrhizic acid, isopropylmethylphenol, hinokitiol, Swertiajaponica extracts, Capsicum tincture, vitamins including, for example,vitamin A, vitamin B, vitamin C, vitamin D, vitamin E, vitamin F,vitamin H, vitamin K, vitamin P, and vitamin U, pantothenyl alcohol,carnitine, ferulic acid, γ-oryzanol, lipoic acid, orotic acid, orderivatives thereof. These compounds are mixed in an amount ranging from0.005 to 10% by mass or, preferably, 0.01 to 2.0% by mass, into theepithelial cell growth promoter of the present invention.

It is optional according to desire that the epithelial cell growthpromoter of the present invention is further admixed with additivesconventionally used in usual lotions such as flavors, coloring agents,pH regulators, disinfectants, surfactants, propellants and the like. Theamount of these additives ranges from 0.001 to 5% by mass or,preferably, 0.01 to 2.0% by mass.

The epithelial cell growth promoter of the present invention is used byrepeatedly applying approximately once to five times a day to the headskin or to the affected skin.

Considering that the epithelial cell growth promoter of the presentinvention can be effective to dose-dependently promote HBK growth, theoligopeptide contained therein as an active ingredient could beconfirmed to have epithelial cell growth-promoting effect.

Moreover, the oligopeptide of the present invention exhibits selectivegrowth-promoting effect on epithelial cells and is thereforeadvantageous without the possibility of growth of other cells such as,for example, cancer cells.

BRIEF DESCRIPTION OF THE DRAWING

FIG. 1 is an illustrative diagram showing a hair cycle of hair growthand hair loss.

FIG. 2 is a mass spectrum of the tripeptide obtained in Example 1.

FIG. 3 is a mass spectrum of the tripeptide obtained in Example 2.

FIG. 4 is a mass spectrum of the tetrapeptide obtained in Example 3.

FIG. 5 is a mass spectrum of the tetrapeptide obtained in Example 4.

FIG. 6 is a mass spectrum of the pentapeptide obtained in Example 5.

FIG. 7 is a bar graph showing the effect of the IGS obtained in Example7 on HBKs growth.

FIG. 8 is a bar graph showing the effect of the PIG obtained in Example7 on HBKs growth.

FIG. 9 is a bar graph showing the effect of the PIGS obtained in Example7 on HBKs growth.

FIG. 10 is a bar graph showing the effect of the GPIG obtained inExample 7 on HBKs growth.

FIG. 11 includes computer-processed diagrams of photographs taken with adigital camera and showing the shaved areas with the group (B) coatedwith application of the epithelial cell growth promoter obtained inExample 8 and the control group (A) on the 14th day from which the ratioof the hair-regrowth area was calculated as percentages using an imageanalysis software.

FIG. 12 is a bar graph showing the effect of cell growth on the HBKsobtained in Example 9.

FIG. 13 is a bar graph showing the effect of cell growth on theepidermal cells obtained in Example 9.

FIG. 14 is a bar graph showing the effect of cell growth on the dermalfibroblasts obtained in Example 9.

FIG. 15 is a bar graph showing the effect of cell growth on the hairpapilla cells obtained in Example 9.

BEST MODE FOR CARRYING OUT THE INVENTION

In the following, the best mode for carrying out the present inventionwill be described by way of Examples. However, the present invention isnot intended to be limited by them in any way.

EXAMPLE 1

Tetrahydrofuran and dicyclohexylcarbodiimide were used as a solvent anda condensing agent, respectively, to produce benzyl ester ofN-Fmoc-isoleucyl-glycyl-serine by successively reacting benzyl ester ofserine having the hydroxyl group protected with a tert-butyl group,glycine having the α-amino group protected with a9-fluorenylmethoxycarbonyl group (hereinafter, abbreviated to Fmocgroup), and isoleucine having the α-amino group protected with an Fmocgroup in the presence of triethylamine by use of an automaticsolid-phase synthesizer (Model “Syro 2000”, manufactured by MultiSynTechCo.).

After the termination of reaction, the elimination of the protectinggroups and the hydrolysis of the ester were performed by treating thegenerated intermediate with hydrofluoric acid in a mixture solvent ofmethyl alcohol and dioxane (3:1 by volume) and purifying it bychromatography, to obtain racemic isoleucyl-glycyl-serine. In this case,the yield thereof was approximately 46%.

Next, this tripeptide was passed through a C₁₈ column [Model “HP1100”(3.0×250 mm), manufactured by Hewlett-Packard Co.] (the column was usedfor purity analysis in Examples 2 to 4 below), and components adsorbedthereon were eluted at a flow rate of 0.4 ml/minute for 20 minutes witha solution of acetonitrile in a concentration ranging from 0 to 30%containing 0.1% trifluoroacetic acid. As a result, the tripeptide waseluted with a retention time of 11.671 minutes in a purity of 95.87%.

The mass of this tripeptide was analyzed with a MALDI-MS massspectrometer [Model “Dynamo”, manufactured by Thermo BioAnalysis Corp.](the mass spectrometer was used for mass spectrometry in Examples 2 to 4below) to show that the mass (m/z, MH⁺) was m/z 275.339. The result ofthis mass spectrometry is shown in FIG. 2.

EXAMPLE 2

Benzyl ester of glycine, isoleucine having the α-amino group protectedwith an Fmoc group, and proline having the α-amino group protected withan Fmoc group were used to produce racemic prolyl-isoleucyl-glycine bytotally the same procedure as in Example 1. In this case, the yieldthereof was approximately 40%.

Next, this tripeptide was passed through a C₁₈ column, and componentsadsorbed thereon were eluted at a flow rate of 0.4 ml/minute for 20minutes with a solution of acetonitrile in a concentration ranging from0 to 30% containing 0.1% trifluoroacetic acid. As a result, thetripeptide was eluted with a retention time of 14.052 minutes in apurity of 95.93%.

The mass of this tripeptide was analyzed to show that the mass (m/z,MH⁺) was m/z 285.102. The result of this mass spectrometry is shown inFIG. 3.

EXAMPLE 3

Benzyl ester of serine having the hydroxyl group protected with atert-butyl group, glycine having the α-amino group protected with anFmoc group, isoleucine having the α-amino group protected with an Fmocgroup, and proline having the α-amino group protected with an Fmoc groupwere used to produce racemic prolyl-isoleucyl-glycyl-serine (SEQ ID NO:2) by totally the same procedure as in Example 1. In this case, theyield thereof was approximately 54%.

Next, this tetrapeptide was passed through a C₁₈ column, and componentsadsorbed thereon were eluted at a flow rate of 0.4 ml/minute for 20minutes with a solution of acetonitrile in a concentration ranging from0 to 40% containing 0.1% trifluoroacetic acid. As a result, thetetrapeptide was eluted with a retention time of 12.313 minutes in apurity of 95.11%.

The mass of this tetrapeptide was analyzed to show that the mass (m/z,MH⁺) was m/z 373.963. The result of this mass spectrometry is shown inFIG. 4.

EXAMPLE 4

Benzyl ester of glycine, isoleucine having the α-amino group protectedwith an Fmoc group, proline having the α-amino group protected with anFmoc group, and glycine having the α-amino group protected with an Fmocgroup were used to produce racemic glycyl-prolyl-isoleucyl-glycine (SEQID NO: 1) by totally the same procedure as in Example 1. In this case,the yield thereof was approximately 48%.

Next, this tetrapeptide was passed through a C₁₈ column, and componentsadsorbed thereon were eluted at a flow rate of 0.4 ml/minute for 20minutes with a solution of acetonitrile in a concentration ranging from5 to 40% containing 0.1% trifluoroacetic acid. As a result, thetetrapeptide was eluted with a retention time of 11.648 minutes in apurity of 99.60%.

The mass of this tetrapeptide was analyzed to show that the mass (m/z,MH⁺) was m/z 343.986. The result of this mass spectrometry is shown inFIG. 5.

EXAMPLE 5

Tetrahydrofuran and dicyclohexylcarbodiimide were used as a solvent anda condensing agent, respectively, to produce benzyl ester ofN-Fmoc-glycyl-prolyl-isoleucyl-glycyl-serine (SEQ ID NO: 3) bysuccessively reacting benzyl ester of serine having the hydroxyl groupprotected with a tert-butyl group, glycine having the α-amino groupprotected with an Fmoc group, isoleucine having the α-amino groupprotected with an Fmoc group, proline having the α-amino group protectedwith an Fmoc group, and glycine having the α-amino group protected withan Fmoc group in the presence of triethylamine by use of an automaticsolid-phase synthesizer (Model “Syro 2000”, manufactured by MultiSynTechCo.).

After the termination of reaction, the elimination of the protectinggroups and the hydrolysis of the ester were performed by treating thegenerated intermediate with hydrofluoric acid in a mixture solvent ofmethyl alcohol and dioxane (3:1 by volume) and purifying it bychromatography, to give racemic glycyl-prolyl-isoleucyl-glycyl-serine.In this case, the yield thereof was approximately 40%.

Next, this pentapeptide was passed through a C₁₈ column [Model“Discovery C₁₈” (4.6×250 mm), manufactured by Supelco], and componentsadsorbed thereon were eluted at a flow rate of 1.5 ml/minute for 20minutes with a solution of acetonitrile in a concentration ranging from2 to 22% containing 0.1% trifluoroacetic acid. As a result, thepentapeptide was eluted with a retention time of 9.761 minutes in apurity of 96.9%.

The mass of this pentapeptide was analyzed with an LC-MS massspectrometer [LC portion: Model “Agilent 1100 Series”, manufactured byAgilent Technologies, Ltd.; MS portion: Model “Thermofinnigan LCQadvantage (software Xcalibur)”, manufactured by Thermo Electron Corp.]by cation measurement using an electrospray ionization method to showthat the mass (m/z, MH⁺) was m/z 430.1. The result of this massspectrometry is shown in FIG. 6.

EXAMPLE 6

Racemic glycyl-prolyl-isoleucyl-glycyl-threonine (SEQ ID NO: 4) wasproduced by totally the same procedure as in Example 1 except thatbenzyl ester of threonine having the hydroxyl group protected with atert-butyl group was used instead of the benzyl ester of serine havingthe hydroxyl group protected with a tert-butyl group. The mass (m/z,MH⁺) of this pentapeptide was m/z 444.3.

EXAMPLE 7

The epithelial cell growth promoters of Examples 1 to 4 were subjectedto a cell growth test for murine HBKs, respectively. The followingculture medium and test medium were used.

(a) Culture medium DMEM (product code “D-5523”, a product by Sigma) FBS(product code “10100756”, a product by Cansera 10% International Inc.)Penicillin/streptomycin (product code “15140-122”,  1% a product byGIBCO) (b) Test medium MCDB153 (product code “M7403”, a product bySigma) Insulin (product code “16634”, bovine, a product by Sigma) 5μg/ml Apo-transferrin (product code “T1147”, human, 10 μg/ml a productby Sigma) EGF (product code “01-101”, murine, a product by 5 ng/mlUpstate Biotechnology Inc.) BPE (product code “13028-014”, bovinepituitary extract, 35 μg/ml a product by GIBCO) Water-solublehydrocortisone (product code “174-00”, 0.5 μg/ml a product by NACALAITESQUE INC.) Ethanolamine (product code “012-12455”, 100 μM a product ofWako Pure Chemical Industries Ltd.) o-phosphorylethanolamine (productcode “P0503”, 100 μM a product by Sigma)(1) Isolation and Culture of HBKs

The skin of a 5 day-old mouse was cut with a scalpel into strips ofapproximately 2-mm-wide skin slices, which were in turn immersed in asolution prepared by dissolving dispase (lot No. 0101, a product by GODOSHUSEI CO.) at a proportion of 500 U/ml in DMEM (product code “D-5523”,a product by Sigma) containing FBS (product code “10100756”, a productby Cansera International Inc.) in a concentration of 5%, and then keptstanding at 4° C. for 16 hours. Subsequently, the epidermis was ablatedand removed with tweezers from the skin slices to collect only dermistissues. The thus obtained dermis tissues were immersed in PBS (−)(product code “P-4417”, a product by Sigma) and cut into strips withophthalmological scissors. The thus obtained strips were immersed in asolution prepared by dissolving collagenase (lot No. 001014W, a productby Nitta Gelatin Inc.) in a concentration of 0.2% in DMEM containing FBSin a concentration of 5%, and then digested at 37° C. for 1 hour. Aftercentrifugation at 1000 rpm for 5 minutes, the supernatant was removed,and the residue was supplemented with PBS (−) and gently pipetted toprepare a dermis suspension.

For separating dermal fibroblasts and hair bulbs, the dermis suspensionwas kept standing for 15 minutes to precipitate only the hair bulbs. Thehair bulbs obtained by repeating the “standing to precipitation”procedure three times were immersed in a solution prepared bydissolving, in 2.65 mM EDTA aqueous solution, trypsin in a concentrationof 0.25%, and then treated at 37° C. for 5 minutes to prepare adispersion solution of HBKs.

Next, this dispersion solution was centrifuged at 1000 rpm for 5minutes. After this centrifugation, the supernatant was removed. Thethus obtained HBKs were dispersed in a culture medium, then inoculatedinto a collagen-coated 96-well microplate, and cultured at 37° C. in anatmosphere containing 5% CO₂.

(iii) Results

As seen in FIG. 7, the cell growth effects on IGS in concentrations of0.3, 1, 3, 10, 30, and 100 μM were 98, 103, 106, 124, 134, and 133%,respectively, as compared with that on the control. Significantdifference (p<0.01 in all the cases) could be noted when theconcentration was 10 μM or higher.

As seen in FIG. 8, the cell growth effects on PIG in concentrations of0.3, 1, 3, 10, 30, and 100 μM were 101, 103, 113, 132, 131, and 134%,respectively, as compared with that on the control. Significantdifference (3 μM: p<0.05; 10, 30, and 100 μM: p<0.01) could be notedwhen the concentration was 1 μM or higher.

As seen in FIG. 9, the cell growth effects on PIGS in concentrations of0.3, 1, 3, 10, 30, and 100 μM were 104, 103, 108, 110, 127, and 133%,respectively, as compared with that on the control. Significantdifference (3 and 10 μM: p<0.05; 30 and 100 μM: p<0.01) could be notedwhen the concentration was 3 μM or higher.

As seen in FIG. 10, the cell growth effects on GPIG in concentrations of0.3, 1, 3, 10, 30, and 100 μM were 105, 108, 118, 133, 134, and 138%,respectively, as compared with that on the control. Significantdifference (0.3 μM: p<0.05; 1, 3, 10, 30, and 100 μM: p<0.01) could benoted when the concentration was 0.3 μM or higher.

The test was further conducted on epidermal cells, dermal fibroblastsand hair papilla cells to find that the epithelial cell growth promoterof the present invention exhibited the same cell growth-promoting effecton the epidermal cells as that on the HBKs along with no influence onthe dermal fibroblasts and the hair papilla cells.

EXAMPLE 8

A 1 mg portion of the pentapeptide obtained in Example 5 was dissolvedin 300 μl of water for injection (product code “057-00456”, a product byOtsuka Pharmaceutical Co.), to which 200 μl of propylene glycol (productcode “161-05006”, a product by Wako Pure Chemical Industries) and 500 μlof ethyl alcohol (product code “057-00456”, a product by Wako PureChemical Industries) were then added and they were mixed together toprepare an epithelial cell growth promoter in a concentration of 1mg/ml.

(2) Cell Growth Test

(i) Sample Preparation

The isoleucyl-glycyl-serine (hereinafter, abbreviated to IGS),prolyl-isoleucyl-glycine (hereinafter, abbreviated to PIG),prolyl-isoleucyl-glycyl-serine (SEQ ID NO: 2) (hereinafter, abbreviatedto PIGS), and glycyl-prolyl-isoleucyl-glycine (SEQ ID NO: 1)(hereinafter, abbreviated to GPIG) obtained in Examples 1 to 4 wereseparately dissolved in a test media to prepare 6 types of samplesolutions each in a concentration of 0.3 μM, 1 μM, 3 μM, 10 μM, 30 μM,and 100 μM. Only a test medium was used as a control.

(ii) HBK Growth Test

After 24 hours from the inoculation of the cells, the culture solutionswere removed, and the cultured cells were washed with an MCDB153solution. Each of the above-mentioned sample solutions was added to thecultured cell at 100 μl/well and cultured at 37° C. in an atmospherecontaining 5% CO₂.

After 4 days, AlamarBlue reagent (registered trademark, cat. No.DAL1100, lot No. AB083002, a product by Biosource International Inc.)was added at 10 μl/well, and the culture was continued at 37° C. in a 5%CO₂ atmosphere. After culturing for 2 hours, fluorescence intensity(excitation wavelength: 544 nm, measurement wavelength: 590 nm) wasmeasured with a microplate reader (Model “Fluoroskan Ascent FL”,manufactured by Labsystems) to evaluate the number of cells.

Incidentally, the evaluation was performed by calculating the ratios(percentage) of the degrees of cell growth for the peptides obtained inExamples 1 to 4 relative to the degree of cell growth for the controland indicating the cell growth ratios by mean value±standard deviation(n=5). A significant difference test was performed by Dunnett's multiplecomparison test (software “Super ANOVA V. 1.11”, a product by AbacusConcepts). When a significance level was less than 5% (p<0.05), it wasconcluded that there was significant difference. The results with theIGS, the results with the PIG, the results with the PIGS and the resultswith the GPIG obtained in this way are shown as bar graphs in FIG. 7,FIG. 8, FIG. 9, and FIG. 10, respectively.

Ten 7-week-old C3H/He female mice were raised for acclimatization for 1week, and the hair on the dorsal of each mouse in the telogen phase of ahair cycle was shaved with an electric clipper and an electric shaver toprepare experimental animals.

Next, these experimental animals were divided into two groups eachcontaining five mice. The epithelial cell growth promoter was applied at100 μl/mouse once a day to the shaved area of the first group from the3rd day after shaving on, while only a mixture of water for injection,propylene glycol, and ethyl alcohol (3:2:5 by volume) was applied as acontrol to the second group. When pictures of the shaved areas weretaken with a digital camera on the 14th day after application, evidenthair growth promotion was observed in the group (B) with application ofthe epithelial cell growth promoter of the present invention, ascompared with that observed in the control group (A).

Next, these images were computer-processed to calculate with imageanalysis software the ratio of the hair-regrowth area (the number ofpixels of hair-regrowth area/the number of pixels of shaved area) aspercentages. A significant difference test was performed by Student's ttest (software “StatVIEW J-4.02”, a product by Abacus Concepts). When asignificance level was less than 5% (p<0.05), it was concluded thatthere was significant difference.

The results are shown in FIG. 11. In this figure, white portionsrepresent unhaired portions, and black portions represent hairedportions. As seen from the results, the ratio of the hair-regrowth areaof the control group (A) was 36.9±5.7%, whereas the ratio of thehair-regrowth area of the group (B) with application of the epithelialcell growth promoter of the present invention was 66.6±3.5%, to showthat the epithelial cell growth promoter of the present inventionpromoted hair regeneration significantly (p<0.01).

EXAMPLE 9

To confirm the cell selectivity of the oligopeptide obtained in Example5 as an epithelial cell growth promoter, the presence or absence of thecell growth-promoting effect on cells existing in skin tissues, that is,HBKs, epidermal cells, dermal fibroblasts, and hair papilla cells, wasexamined in the following manner. Incidentally, the same culture mediumand test medium as in Example 7 were used.

(1) Preparation of HBKs, Epidermal Cells, Dermal Fibroblasts, and HairPapilla Cells

The skin of a 5 day-old C3H/HeN mouse was aseptically collected and cutwith a surgical knife into samples of strips of approximately 2-mm-wideskin slices. The above-mentioned samples were immersed in a solutionprepared by dissolving 500 U/ml of dispase (lot No. 0101, a product byGODO SHUSEI CO.) in DMEM (product code “D-5523”, a product by Sigma)containing 5% by mass of FBS (lot No. 0101, a product by CanseraInternational Inc.), and then kept standing at 4° C. for 16 hours.Subsequently, epidermis tissues were ablated with tweezers from the skinslices and separated from dermis tissues. The thus obtained epidermistissues were subjected to the separation of epidermal cells, and thedermis tissues were subjected to the separation of HBKs and dermalfibroblasts.

The thus obtained dermis tissues were immersed in PBS (−) (product code“P-4417”, a product by Sigma) and cut into strips with ophthalmologicalscissors. The strips were immersed into a solution prepared bydissolving 0.2% by mass of collagenase (lot No. 001014W, a product byNitta Gelatin Inc.) in DMEM containing 5% by mass of FBS, and thendigested at 37° C. for 1 hour. The thus obtained dermis digestionsolution was centrifuged at 1000 rpm for 5 minutes. The supernatant wasremoved, and the residue was supplemented with PBS (−) and gentlypipetted to prepare a dermis suspension.

For separating dermal fibroblasts and hair bulbs, this dermis suspensionwas kept standing for 15 minutes to precipitate only the hair bulbs. The“standing to precipitation” procedure was repeated three times. The thusobtained hair bulbs were immersed in a solution preparing by dissolving0.25% by mass of trypsin in 2.65 mM aqueous EDTA solution, and thentreated at 37° C. for 5 minutes to prepare a dispersion solution ofHBKs.

Next, this dispersion solution was centrifuged at 1000 rpm for 5minutes. After this centrifugation, the supernatant was removed. Thethus obtained HBKs were dispersed in a culture medium, then inoculatedinto a collagen-coated 96-well microplate, and cultured at 37° C. in anatmosphere containing 5% CO₂.

On the other hand, dermal fibroblasts floating in the supernatant by thefirst “standing to precipitation” procedure were collected andcentrifuged at 1000 rpm for 5 minutes. After this centrifugation, thesupernatant was removed. The thus obtained dermal fibroblasts weredispersed in a culture medium, then inoculated into a 96-wellmicroplate, and cultured at 37° C. in an atmosphere containing 5% CO₂.

The above-mentioned epidermis tissues were immersed in a solutionprepared by dissolving 0.25% by mass of trypsin in 2.65 mM aqueous EDTAsolution, and then treated at 37° C. for 5 minutes to prepare adispersion solution of epidermal cells.

Subsequently, this dispersion solution was centrifuged at 1000 rpm for 5minutes, and the supernatant was removed. The thus obtained epidermalcells were dispersed in a culture medium, then inoculated into acollagen-coated 96-well microplate, and cultured at 37° C. in anatmosphere containing 5% CO₂.

Moreover, human hair papilla cells (product code “602-05”, a product byTOYOBO CO.) were dispersed in a culture medium, then inoculated into acollagen-coated 96-well microplate, and cultured at 37° C. in anatmosphere containing 5% CO₂.

(2) Cell Growth Test

(i) Sample Preparation

A 1 mg portion of the pentapeptide obtained in Example 5 was added to232.8 μl of a test medium and dissolved by stirring to prepare a 10 mMsolution. Subsequently, this 10 mM solution was stepwise diluted toprepare 6 types of sample solutions each in a concentration of 100 μM(No. 1), 30 μM (No. 2), 10 μM (No. 3), 3 μM (No. 4), 1 μM (No. 5), and0.3 μM (No. 6). Only a test medium was used as a control.

(ii) HBK and Epidermal Cell Growth Tests

After 24 hours from the inoculation of the cells, the culture solutionswere removed, and the cultured cells were washed with an MCDB153solution. Each of the above-mentioned sample solutions was added to thecultured cell at 100 μl/well and cultured at 37° C. in an atmospherecontaining 5% CO₂. After 4 days, AlamarBlue reagent (registeredtrademark, cat. No. DAL1100, lot No. AB083002, a product by BiosourceInternational Inc.) was added at 10 μl/well, and the culture wascontinued at 37° C. in an atmosphere containing 5% CO₂. After 2 hours,fluorescence intensity (excitation wavelength: 544 nm, measurementwavelength: 590 nm) was measured with a microplate reader (Model“Fluoroskan Ascent FL”, manufactured by Labsystems) to evaluate thenumber of cells. A significant difference test was performed byDunnett's multiple comparison test (software “Super ANOVA V. 1.11”, aproduct by Abacus Concepts). When a significance level did not exceed 5%(p<0.05), it was concluded that there was significant difference.

The results of the HBK growth tests and the results of the epidermalcell growth tests obtained in this way are each shown as bar graphs inFIG. 12 and FIG. 13.

(iii) Dermal Fibroblast and Hair Papilla Cell Growth Tests

After 24 hours from the inoculation of the cells, the culture solutionswere removed, and the cultured cells were washed with a DMEM solution.Each of the above-mentioned sample solutions was added to the culturedcell at 100 μl/well and cultured at 37° C. in an atmosphere containing5% CO₂. Moreover, culturing was performed in the same manner with aculture medium as a positive control. After 3 days, AlamarBlue reagent(product code “341-077612”, a product by Biosource International Inc.)was added at 10 μl/well, and the culture was further continued at 37° C.in an atmosphere containing 5% CO₂. After 2 hours, fluorescenceintensity (excitation wavelength: 544 nm, measurement wavelength: 590nm) was measured with a microplate reader (Model “Fluoroskan Ascent FL”,manufactured by Labsystems) to evaluate the number of cells.

The results of the dermal fibroblast growth tests and the results of thehair papilla cell growth tests obtained in this way are each shown asbar graphs in FIG. 14 and FIG. 15.

(iv) Results

As seen in FIG. 12, the effects of the sample solutions on the HBKgrowth were 101% (No. 6), 111% (No. 5), 124% (No. 4), 141% (No. 3), 142%(No. 2), and 141% (No. 1), respectively, as compared with that of thecontrol. Significant difference (p<0.01) could be noted when theconcentration was 1 μM or higher.

This demonstrated that the epithelial cell growth promoter of Example 5dose-dependently exhibited the HBK growth-promoting effect in aconcentration of 1 μM or higher.

As seen in FIG. 13, the effects of the sample solutions on the epidermalcells were 102% (No. 6), 110% (No. 5), 118% (No. 4), 124% (No. 3), 127%(No. 2), and 127% (No. 1), respectively, as compared with that of thecontrol. Significant difference (1 μM: p<0.05, 3 μM or higher: p<0.01)could be noted when the concentration was 1 μM or higher.

This demonstrated that the epithelial cell growth promoter of Example 5dose-dependently exhibited the growth-promoting effect on epidermalcells as well as HBKs in a concentration of 1 μM or higher.

On the other hand, as seen in FIGS. 14 and 15, the epithelial cellgrowth promoter of Example 5 was shown to have no influence on dermalfibroblasts and hair papilla cells.

These facts demonstrated that the epithelial cell growth promoter ofExample 5 exhibited selective cell growth-promoting effect on HBKs andepidermal cells classified into epithelial cells.

In this way, the epithelial cell growth promoter of the presentinvention exhibits selective cell growth-promoting effect on HBKs andepidermal cells classified into epithelial cells so as not adversely toaffect cells other than epithelial cells constituting skin tissues, whenused as a liniment such as, for example a lotion.

INDUSTRIAL APPLICABILITY

The compound of the present invention can be employed as an epithelialcell growth promoter for hair growth and skin regeneration. According tothe present invention, a novel excellent epithelial cell growth promoterhaving not only hair growth effect but also skin regeneration effect andtherapeutic effect on atopic dermatitis is provided.

1. A water-soluble peptide consisting ofglycyl-prolyl-isoleucyl-glycyl-serine [SEQ ID NO: 3] orglycyl-prolyl-isoleucyl-glycyl-threonine [SEQ ID NO: 4], or awater-soluble salt thereof.
 2. The water-soluble peptide according toclaim 1, wherein the peptide is glycyl-prolyl-isoleucyl-glycyl-serine[SEQ ID NO: 3], or a water-soluble salt thereof.
 3. The water-solublepeptide according to claim 1, wherein the peptide isglycyl-prolyl-isoleucyl-glycyl-threonine [SEQ ID NO: 4], or awater-soluble salt thereof.
 4. A composition comprising thewater-soluble peptide according to claim 1, or a water-soluble saltthereof, in combination with a carrier.